Functional Analysis Of AP2/ERF Family Transcription Factors BnaERF-B In Brassica Napus

Plants defend themselves from different sources of biotic and abiotic stress by various defense mechanisms. One of the most rapid and effective defense responses is plant signal regulation by transcription factors. AP2/ERF factors play very important roles in plant development, hormonal regulation and stress response.Here, we cloned an AP2/ERF family transcription factor gene BnaERF-B3-hy15 from a winter type B. napus L. cv HuYou15. Real-time RT-PCR analyses were performed to determine the expression profiles of BnaERF-B3-hy15. The expression of BnaERF-B3-hy15 was induced by dehydration (PEG), high salt (200mM NaCl), and ABA (200μM), and cold (4℃) in three-week-old seedlings. In addition, we examined tissue-specific expression of BnaERF-B3-hy15 in HuYou15 RT-PCR. The expression of BnaERF-B3-hy15 in B. napus L. cv HuYou15 at the period of flowering and seed development was detected in root and pod, and none or weak expressed in the bud, petal, leaf and stem.The BnaERF-B3-hy15 factor has only weak GCC box-binding activity by the yeast one-hybrid system for investigation. We mutant the BnaERF-B3-hy15 gene and got a mutant, named BnaERF-B3-hy15-mu3, which exhibited more binding activity with the GCC box element than the wild-type factor BnaERF-B3-hy15. The transgenic Arabidopsis plants over-expressed the mutant BnaERF-B3-hy15-mu3 gene exhibited more freezing tolerance than transgenic plants contained original BnaERF-B3-hy15 gene. In contrast to non-transgenic plants, these transgenic lines showed hypersensitive to ABA and may be involved in ABA-dependent pathway. Real-time PCR analyses revealed that the expression levels of several cold-regulated genes were altered in the over-expressed BnaERF-B3-hy15-mu3 transgenic lines. In order to confirm this hypothesis, gene expression analysis of possible regulated genes located down-stream in transgenic plants was carried out by real-time RT-PCR. The gene cor47, cor15a, rd29b, rd29a was up-regulated in BnaERF-B3-hy15-mu3 transgenic lines under cold stress.It is reasonable to presume that BnaERF-B3-hy15-mu3 participates in ABA-dependent signaling transducation pathway and can enhance cold tolerances for transgenic Arabidopsis based on research results.